The long-term goal of the proposed research is to elucidate the molecular details of preprotein targeting to and translocation across biological membranes utilizing Escherichia coli as a facile genetic and biochemical system suitable for structural analysis. The project will specifically focus on a central component of this system, SecA ATPase, which binds both preproteins and the SecYEG channel complex, and whose translocation ATPase activity and membrane insertion and retraction cycle are at the heart of the protein translocation mechanism. Three specific aims are proposed. (1) To resolve the controversy surrounding the location and topology of the SecA signal peptide-binding site, a series of chimeras with E. coli SecA attached to two different signal peptides will be constructed and tested for signal peptide binding and transport function. Functionally-optimized chimeras will be re-engineered by substitution with B. subtilis or T. maritima SecA, retested and crystallized in order to determine their x- ray structures. These studies should define the molecular basis of signal peptide binding to SecA, and they should also help to reveal how the signal peptide is inserted into the SecYEG channel. (2) To reconcile the SecA membrane insertion-retraction model with recent structural studies of the SecYEG complex that limit channel size as well as to characterize the integral membrane structural state of SecA at SecYEG, an in vivo and in vitro site-specific photocrosslinking approach will be utilized to map SecY-interaction sites with this form of SecA deep within the translocon complex during normal or translocation-arrested conditions. These studies should help to define the poorly understood structural state of integral membrane SecA and allow for a re-evaluation of this dominant model. (3) To resolve the controversy as to whether SecA functions as a monomer or dimer and define any relevant dimer state at the active translocon, an in vivo and in vitro site-specific photocrosslinking approach wil be undertaken to identify and characterize SecYEG-bound SecA dimer during normal or translocation- arrested conditions. Use of photocrosslinkable signature residues unique to each SecA dimer interface will allow discrimination between the different SecA dimer states. These studies should clarify the oligomeric state(s) of SecA at the active translocon and allow more definitive models of SecA action to be proposed and tested. Overall, these studies will allow a better understanding of Sec-dependent protein transport at the molecular level in three critical unresolved areas of SecA structure and function, and they should be of broad significance to ultimately engineer this pathway for secretion of biopharmaceuticals and develop novel anti-bacterial agents to its conserved components in human pathogens.